Chemical amplification, the formation of an enhanced chemical response, occurs in three ways. The first is catalysis, a common example of which is the action of an enzyme or coenzyme. Enzyme or substrate cycling (1), in which a substrate acts as a catalyst by first participating in one reaction, and then cycling back to its original state in a second reaction, also illustrates catalytic amplification. In gate amplification, the opening of a molecular gate such as a channel in a membrane amplifies the passage of molecules from one zone to another. Ultimately complement, for example, has such an effect on a target cell. Third, there is multiplicative amplification, in which the amount of a substance is multiplied by a constant factor repetitively, e.g., the unhindered successive generations of a virus. These three mechanisms of amplification also can occur combined. For example, in the overall action of immune complement, catalysis and gate mechanisms are both present.
The role of chemical amplification in chemical analysis, including clinical chemistry, was carefully reviewed by Blaedel and Boguslaski in 1978 (2). Amplification components such as enzyme, coenzymes, inorganic iodide, catalytic electrodes, bacteriophage and liposomes were all discussed. When such components inherently don't provide the specificity required, or don't directly involve the analyte, then an antibody, secondary enzyme, or secondary chemical reaction is used to make the amplification system specific or analyte-responsive. Aside from the simple use of enzymes as amplification catalysts, many of the techniques discussed by these authors for clinical analysis were not very practical at that time. The most general problem was the tendency of these techniques to require several complex, highly-purified reagents, leading to the secondary difficulties of short shelf-life, high cost, and assay irreproducibility. The techniques were also tedious to set up or use, and gave a limited or slow rate of amplification.
A more recent example of gate amplification in chemical analysis is the report by Litchfield et al. (3) of an immunoassay for digoxin in which ouabainmellitin triggers the release of alkaline phosphatase entrapped in a liposome. This approach to chemical amplification has many disadvantages: liposomes tend to be unstable; a threshold dose of a mellitin substance is required and probably acts only once to release alkaline phosphatase; mellitin is a hydrophobic substance that will tend to behave eratically be undergoing losses onto surfaces and forming complexes with interfering macromolecules; and the behavior of liposomes in an actual assay can be influenced by other substances in the sample that bind to the liposome aside from the ouabain-mellitin reagent.
Stanley et al (4), have extended substrate cycling by incorporating a color reaction (formation of a formazan dye) directly into a NAD/NADH cycle. A cycling time of about 50 min.sup.-1 was reported, and the system was used in an enzyme immunoassy for thyroid stimulating hormone. However, this is not a very high rate of chemical amplification. Other disadvantages of this system are as follows: the reagents involved are expensive, especially because they must be provided in a highly purified form and some of them are not commonly used; the system is not very flexible in terms of its components; physiological samples tend to contain enzymes that utilize NAD/NADH and therefore can interfere with the assay; the system is unable to yield other signals beside a color rejection tied to NAD/NADH; three different enzymes are used, making it impossible to find conditions that are simultaneously optimum for each, and increasing the likelihood that a given sample will contain an inhibitor for one of these enzymes, leading to faulty results; and NADH is not a stable substance.
Harris (U.S. Pat. No. 4,463,090) has used cascade amplification in an enzyme immunoassay in which one or more proenzymes are present. In this invention, an initial enzyme or activator begins a sequence of activating a proenzyme to an enzyme, which enzyme product may be detected or may activate another proenzyme to an enzyme that may be detected, or it may in turn activate another proenzyme etc. Thus there is a cascade of reactions in which different enzymatic activities sequentially are activated. The Harris invention uses naturally occurring enzyme cascades that unfortunately are limited in number. It comprises components that tend to be unstable, susceptible to inhibitors, complex, not readily available, poorly characterized and expensive. Such reagents tend to vary in their properties batch-to-batch. This is especially true for the enzyme components, and these problems are multiplied because two or more such enzymes are used in each system. Thus this approach to chemical amplification is not particularly practical. Consistent with this, no experimental work is cited in this invention. A model ligand assay using two proenzymes from the blood-coagulation cascade has been reported (D. A. Blake, M. T. Skarstedt, J. L. Shutlz and D. P. Wilson, Clin. Chem. 30, [1984] 1452-1456), but the degree of amplification was low, the assay was not very sensitive, and the reproducibility of the system was not evaluated.
Thus, for chemical amplification in chemical analysis, not much progress has been made since 1978 when Blaedel and Boguslaski, as cited above (reference 2), concluded that the systems available then were not very practical. Beyond the simple use of enzymes as inherent amplification catalysts, no systems have been adopted for general use because of these problems. The main limitation of a simple enzyme as an inherent catalyst is the limited amplification this provides, since an enzyme by itself only produces a constant amount of product molecules from substrate per unit time.